The mitochondrial genome is vital forCaenorhabditis elegansmetabolism, physiology, and development. TheC. elegansmitochondrial DNA is typical of animal mitochondrial genomes in its size and gene content. It is 13,794 nucleotides in length
and encodes 36 genes: 2 ribosomal RNAs, 22 transfer RNAs, and 12 protein subunits of the mitochondrial respiratory chain.
Although it represents only a small number of genes, an elaborate cellular machinery comprised of over 200 nuclear genes is
needed to replicate, transcribe, and maintain the mitochondrial chromosome and to assemble the translation machinery needed
to express this dozen proteins. Mitochondrial genetics is peculiar and complex because mitochondrial DNA is maternally inherited
and can be present at tens to tens of thousands of copies per cell. The mitochondrial genome content of the developing nematode
is developmentally regulated; it increases about 30-fold between the L1 and the adult stages and blocking the increase leads
to larval arrest. Energy metabolism is also intimately linked to aging and lifespan determination. The nematode model system
offers numerous advantages for understanding the full importance and scope of the mitochondrial genome in animal life.

1. Introduction

The mitochondrial genome is indispensable to the cellular and organismal biology of Caenorhabditis elegans. An elaborate cellular machinery is
employed to maintain mitochondrial DNA (mtDNA), to express it, and to ensure its
inheritance. Because it is maternally transmitted and because of its wide-ranging copy
number (it can be present at fewer than 100 to tens of thousands of copies per cell),
the genetics of mtDNA are characterized by a number of peculiar and as yet incompletely
understood features. The fundamental importance of mtDNA to cellular energy metabolism
explains the profound effects mtDNA mutations can have; mitochondria are the major
source of reactive oxygen species, which are implicated in premature aging and senescence.

Mitochondria play a pivotal role in cellular metabolism. They were once free-living
organisms related to modern eubacteria and they continue to perform many of the
biochemical and physiological functions of their bacterial ancestors (Timmis et al., 2004). Mitochondria are double-membrane organelles
most commonly associated with oxidative phosphorylation, a process that meets the
majority of cellular energy demands. In addition, mitochondria are involved in heme,
lipid, nucleotide, iron-sulfur cluster, and amino acid biosynthesis: they are home to
the citric acid cycle, the urea cycle and fatty acid oxidation.

2. Mitochondrial DNA structure

Mitochondria harbor a small but essential component of an eukaryote's genetic
material. The symbiotic relationship that marks the origin of eukaryotes was established
approximately 2 billion years ago and has been followed by a massive loss or transfer of
genes to the host genome; only a tiny fraction (less than one percent) of the
endosymbiont's genome is retained on the mtDNA (Gray et al., 2001). Animal mtDNAs are extremely compact, being less than 20
kilobases in length, and encode fewer than 40 genes (Wolstenholme, 1992). All of the proteins encoded by the mtDNA are subunits of the
mitochondrial respiratory chain (MRC; Okimoto et al., 1992). Despite their small number, the genes on the mtDNA necessitate a
large expenditure of cellular resources because they are essential for strict aerobes.
Hundreds of nuclear genes are needed to replicate, transcribe, and maintain the
mitochondrial chromosome and to assemble the translation machinery needed to express its
dozen or so proteins (Tsang and Lemire, 2003).

The C. elegans mtDNA is 13,794 nucleotides in length and encodes
36 genes: 2 ribosomal RNAs (12S rRNA and 16S rRNA), 22 transfer RNAs, and 12 MRC
subunits (Figure 1; Okimoto et al., 1992).
A typical animal mtDNA, the nematode mitochondrial genome is slightly smaller than its human counterpart.
It lacks the ATP8 gene of the human genome, which encodes a subunit of the ATP synthase (complex V). Introns are absent and
there are few or no non-coding nucleotides between
genes; approximately 92% of the mitochondrial genome has coding function. Three genes
end with incomplete termination codons (T or TA) that are apparently converted to TAA
codons by polyadenylation of the transcript. Only one sizeable non-coding region called
the displacement loop or D-loop is present. The replication and transcription of mtDNA
has been mainly studied in mammals where the D-loop region contains one of the origins
of replication and the promoters for mtDNA transcription (Garesse and Vallejo, 2001). Few components needed for the maintenance or
expression of the C. elegans mtDNA have been experimentally
identified. The CLK-1 (biological clock abnormal) protein was reported to have DNA binding activity specific for the light strand origin of replication
(OL) on the mtDNA,
suggesting a role in regulating mtDNA replication or transcription in C.
elegans (González-Halphen et al., 2004; Gorbunova and Seluanov, 2002).

Figure 1. Gene map of the C. elegans mtDNA. The molecule contains the genes for twelve proteins (thick grey arrows), two rRNAs
(black arrows), and 22 tRNAs (circles labeled with one-letter amino acid code). The
serine and leucine tRNAs are also identified by the codon family recognized. The
positions of the putative D-loop and of the uaDf5 deletion mutation are indicated inside and outside the circle, respectively.

In contrast to the relatively uniform gene content of metazoan mtDNAs, the
mitochondrial genetic codes are highly modified. In C. elegans, TGA
specifies tryptophan rather than being a stop codon, ATA specifies methionine rather
than iso-leucine, and AGA and AGG specify serine rather than arginine. The translation
initiation codon ATG is not used; ATT, ATA, or TTG codons apparently serve as initiation
codons (Okimoto et al., 1992; Okimoto et al., 1990). These peculiarities of the genetic code may in
part explain the retention of the twelve protein-coding genes on the mtDNA rather than
their transfer to the nucleus.

3. mtDNA-encoded proteins

The C. elegans mtDNA encodes 12 MRC subunits out of a total of
the approximately 80 subunits assembled into five MRC complexes (Figure 2). Four of these five complexes contain mtDNA-encoded subunits; complex II, the
succinate-ubiquinone oxidoreductase complex, is the exception. The ND1, ND2, ND3, ND4,
ND4L, ND5, and ND6 subunits of complex I (the NADH-ubiquinone oxidoreductase) are all
core subunits localized in the membrane arm of the enzyme. The ND1 subunit is implicated
in ubiquinone binding (Schultz and Chan, 2001). The
cytochrome b of complex III (the ubiquinol-cytochrome c
oxidoreductase) is encoded by the ctb-1 gene in C.
elegans; it is an essential component of the enzyme, harboring 2
b-type hemes. Similarly, the COI, COII, and COIII subunits,
which coordinate catalytic heme and copper cofactors, are essential components of
complex IV (the cytochrome c oxidase). Finally, the ATP6 subunit of complex V plays a vital role in the efficiency of ATP synthesis (Lenaz et al., 2004). Not surprisingly, mutations in each of the human
orthologs are causal in a variety of neurological, endocrinological, and muscular
diseases (DiMauro, 2004).

Very few mutations in the nematode mtDNA have been reported. In contrast, over 100
point mutations and 200 insertions, deletions, or rearrangements have been described for
the human mtDNA (see MITOMAP: A Human Mitochondrial Genome Database). It is puzzling why mtDNA mutations have not been reported in C. elegans, given the large number of genetic screens for defects in motility, reproduction, development, and other functions. Perhaps
the nematode can better tolerate deleterious mtDNA mutations or has mechanisms to prevent their transmission. The genetics
of the mitochondrial genome still offer much to
be explored.

4. Mitochondrial DNA inheritance

It is generally believed that animal mtDNA is inherited exclusively from the mother.
However, it was recently shown in a patient with mitochondrial myopathy that the
pathogenic mtDNA was of paternal origin (Schwartz and Vissing, 2002). Evidence for the occasional paternal transmission of mtDNA has
also been reported in sheep, mice, cattle, mussels, and insects (Zhao et al., 2004). Although rare, the paternal inheritance of mtDNA
may have a significant impact on evolution and on disease development.

5. Mitochondrial dynamics

Mitochondria are dynamic structures and organelle plasticity is related to mtDNA
inheritance (Garrido et al., 2003). Multiple mtDNA
molecules are organized into discrete protein-DNA complexes called nucleoids
(Jacobs et al., 2000). Mitochondria form
reticular or tubular networks and their interaction with cytoskeletal components
provides clues to their distribution, movement, and inheritance (Rube and van der Bliek, 2004). A mutation in the
anc-1 (anchorage of nuclei abnormal) gene of C.
elegans resulted in hypodermal nuclei and mitochondria that float freely in
the cytoplasm of syncytial cells (Hedgecock and Thomson, 1982). These mitochondria adopted an almost spherical shape and were often
clustered. ANC-1 contains several coiled coil regions, a nuclear envelope localization
domain, and an actin-binding domain, suggesting it mediates connections from nuclei and
mitochondria to the actin network and to the nuclear envelope (Starr and Han, 2002). Mitochondrial division is required to regulate
organelle numbers during cell division, differentiation, and in response to
environmental conditions. The C. elegansDRP-1, a dynamin-related
protein with a characteristic GTPase domain, plays a key role in mitochondrial
distribution and division (Labrousse et al., 1999).
RNAi of drp-1 results in embryonic lethality and abnormal
mitochondrial morphology and distribution, while overexpression leads to fragmentation
of the organelle. DRP-1 is involved in the fission of the mitochondrial outer membrane,
perhaps directly or perhaps through its ability to recruit additional fission machinery
components.

6. mtDNA copy number

The numbers of mitochondria and mitochondrial genomes in a cell are regulated and can
vary substantially between tissues (Moraes, 2001).
The developmental regulation of mtDNA copy numbers in C. elegans
has been investigated (Tsang and Lemire, 2002). An
embryo contains ∼25,000 copies of mtDNA and this number remains unchanged through the
L1, L2, and L3 larval stages. A fivefold increase to 1.3 x
105 copies in the L4 stage is followed by a further sixfold
increase to 7.8 x 105 copies in the adult hermaphrodite. The
first copy number increase coincides with sexual maturation; spermatogenesis begins in
the early L4 stage and oogenesis in the young adult.

mtDNA copy number is closely tied to reproduction. Mutations in the
glp-1 gene, which is involved in germ line proliferation, lead to
an almost complete absence of germ line cells while somatic cell numbers are normal
(Austin and Kimble, 1987). The mtDNA content of
an L4-stage glp-1 hermaphrodite is ∼70,000, half of wild type
levels (Tsang and Lemire, 2002). There is no further
increase in mtDNA copy number as the glp-1 animal matures. Thus,
increases in mtDNA content from the L3 to the adult stages can be subdivided into two
components: a somatic component is glp-1 independent and occurs
during development from the L3 (∼25,000 copies) to the L4 stage (∼70,000 in the
glp-1 L4) and a germ line component accounts for the remainder
of the increase to 7.8 x105 in the adult.

Oocyte production accounts for the majority of the germ line-related increases in
mtDNA copy numbers. A loss of function mutation in the feminization gene
fem-1 blocks sperm production in the hermaphrodite but does not
affect mtDNA contents in the L4 or in the adult (Tsang and Lemire, 2002). In contrast, a gain of function mutation in the
fem-3 gene, which blocks oocyte production, does not affect L4
mtDNA content but reduces the content in the adult to about one quarter (to 1.9 x
105 copies) of wild type levels. Thus, the majority of
organellar genomes are associated with oocyte development and the sperm-associated
component is minor. When determined independently, 18,000 copies of mtDNA were measured
per fem-1 oocyte, a number similar to wild type embryos and early
larvae.

The mtDNA content of individual C. elegans cells is considerably
lower than the estimated values of 1,000-10,000 copies per cell in higher eukaryotes
(Tsang and Lemire, 2002). The ∼25,000 copies of
mtDNA in the L1 larva, if evenly distributed around the 558 nuclei, suggest an average
of 45 copies per cell. Similarly, the L4 larva has ∼1,000 somatic nuclei and 70,000
copies of mtDNA in a glp-1 mutant, corresponding to 70 copies per
cell. It remains to be determined whether different somatic cells or tissues have
distinct mitochondrial genome contents that might reflect their energy requirements.
Estimated cellular contents of mtDNA for undifferentiated germ line cells and for sperm
are 250 and 30-40, respectively. The increases in mtDNA copy numbers during development
are suggestive of parallel increases in the numbers of organelles, although this has not
been documented. Interestingly, there is an increase in organelle number and in the
frequency of organelle division with a shift of temperature from 15 to 25°C (Labrousse et al., 1999). This increased content of mitochondria may reflect an adaptation
to the higher metabolic demands of elevated temperatures. Consistent with this notion,
respiration rates (Van Voorhies and Ward, 1999) and
ubiquinone levels (Jonassen et al., 2002) are
doubled with similar temperature shifts.

7. Role of mtDNA in development

A functional MRC is essential for viability and larval development past the L3 stage.
Mutations in nuclear genes encoding MRC subunits such as nuo-1 or
atp-2 (subunits of complexes I and V respectively) lead to a
characteristic L3 stage arrest having an L2-staged gonad (Tsang and Lemire, 2003; Tsang et al., 2001). Similarly, clk-1 mutations, which impair
ubiquinone biosynthesis, can lead to an L2 or L3-stage arrest (Hihi et al., 2002; Jonassen et al., 2001). Inhibitors of mtDNA replication or transcription such as ethidium
bromide or inhibitors of mitochondrial translation such as chloramphenicol or
doxycycline can also quantitatively produce an L3 stage developmental arrest
(Tsang and Lemire, 2002; Tsang et al., 2001). Maturation to the L4 stage likely entails increased
energy demands that are met with de novo synthesis of new mitochondrial energy
production capacity. An energy sensor that responds to the concentrations of one or more
metabolites, such as ATP or NADH, may communicate information about the status of
mitochondrial energy generation and lead to altered patterns of MRC gene expression
(Tsang and Lemire, 2003; Tsang et al., 2001).

8. mtDNA mutations

Mitochondrial genetics differ from nuclear genetics in three aspects. First,
mitochondrial genes are maternally inherited; they do not follow a Mendelian pattern of
inheritance. Second, the mitochondrial genome is polyploid. Normally, a state of
homoplasmy exists where only one form of mtDNA is present. Mutation can lead to a state
of heteroplasmy where two or more forms of mtDNA coexist within a cell. Third, unlike a
diploid nuclear gene that can normally only assume three states (homozygous wild type,
heterozygous, or homozygous mutant), mtDNA heteroplasmy does not vary by discrete steps.
The proportions of mtDNA species can vary with time or through mitotic segregation as
cells divide.

The inheritance and maintenance of mtDNA species are most poorly understood in the
heteroplasmic condition. The uaDf5 mtDNA deletion, which removes 11
genes (four MRC subunit and seven tRNA genes), is maternally inherited and stably
propagated without selection (Figure 1; Tsang and Lemire, 2002). While individual
animals have uaDf5 mtDNA contents ranging from ∼20% to ∼80%, the
population average is ∼60% and this value does not vary between larval stages. There is
no phenotype associated with the deletion, even in animals with an ∼80% content of
uaDf5 mtDNA. Most surprisingly, homoplasmic wild type or mutant
animals have not been detected in over 100 generations, suggesting the two forms of
mtDNA do not segregate from each other. Single, self-fertilized hermaphrodites with
intermediate levels of uaDf5 mtDNA (∼50-60%) have offspring
containing from 20-80% mutant mtDNA, indicating that the level of heteroplasmy can
change significantly in one generation. However, hermaphrodites with extreme levels of
heteroplasmy (∼20% or ∼80%) only produce offspring with more moderate levels, suggesting
two opposing forces are in operation. One force increases uaDf5
contents when they are low, while the second decreases them when they are high. One
possible explanation for this phenomenon is that wild type mtDNA contains an undetected
mutation that is complemented by the uaDf5 mtDNA. Thus, both
genomes are needed to produce a fully functional MRC and homoplasmy for either mutation
would be severely compromising or lethal. Alternatively, the stable heteroplasmy may
result from intra-mitochondrial heteroplasmy, a condition where the mtDNA species are
intermixed within the nucleoid of one organelle (Jacobs et al., 2000).

Although the heteroplasmic uaDf5 animals do not have a phenotype,
they have adjusted their mtDNA contents. Both hermaphrodites and males have
approximately twice the number of mitochondrial genomes as their wild type counterparts
(Tsang and Lemire, 2002). The upregulation of
mtDNA copy number may in part compensate for the effects of the mutation by enhancing
the production of functional mitochondrial transcripts and/or proteins. Understanding
the signals and mechanisms employed to regulate mtDNA copy number remains an important
challenge.

The C. elegans mtDNA mutation rate has been estimated using a
series of mutation accumulation lines maintained by single-progeny descent for over 200
generations (Denver et al., 2000). In over 770,000
base pairs of sequenced DNA, 16 base substitution and 10 insertion/deletion mutations
were detected, corresponding to a measured rate approximately 100-fold higher than
previous indirect estimates. Four of the insertion/deletion mutations introduced drastic
changes to coding sequences, were homoplasmic (or nearly so), but had only modest
effects on fitness. Most spontaneous mtDNA mutations in C. elegans
appear to be mildly deleterious although selection may occur in a natural context.

The mtDNA mutation rate can be increased by environmental agents or by mutation of
nuclear genes involved in mtDNA maintenance. For example, nucleoside analogs used in the
clinical treatment of viral infections, such as human immunodeficiency virus, inhibit
the mtDNA polymerase, which is essential for mtDNA synthesis and repair
(Martin et al., 2003). A mutation that impairs
the fidelity of the mtDNA polymerase produced a mtDNA mutator phenotype in a mouse
model; this was associated with premature aging and reduced lifespan
(Trifunovic et al., 2004).

9. mtDNA and aging

Mitochondrial functions influence and possibly control the rate of aging
(Dillin et al., 2002) and conversely, aging
affects the integrity of the mitochondrial genome. An increased production of reactive
oxygen species, often associated with MRC dysfunction, can precipitate premature aging,
cause mutations in the mitochondrial and nuclear genomes, and shorten lifespan
(Hartman et al., 2004). The first C.
elegans mtDNA deletions detected were spontaneous events in an aging,
wild type population (Melov et al., 1994;
Melov et al., 1995). age-1
(aging abnormal) mutants, which are more resistant to oxidative stress and live twice
as long as wild type, have a lower frequency of mtDNA deletions (Melov et al., 1995). It has not been determined whether these
aging-associated mtDNA deletions reduce fitness or whether they are heritable.

To date, there is no direct evidence linking genes encoded by mtDNA with aging, but
mutations in nuclear genes provide ample reason to believe that they are involved. The
isp-1(qm150) mutation, which affects the iron sulfur protein of
complex III, was the first mutation of an MRC subunit reported to increase lifespan
(Feng et al., 2001). The mtDNA-localized,
maternally-inherited ctb-1(qm189) mutation affecting the cytochrome
b subunit of complex III, does not alter the long life span of
qm150 animals but does partially suppress other effects of the
qm150 mutation (Feng et al., 2001). The ctb-1(qm189) mutation has no independent
phenotype. The genetic interaction of the isp-1 and
ctb-1 mutations clearly illustrates the interplay of the
mitochondrial and nuclear genomes in cellular metabolism. The
mev-1(kn1) mutation is a missense allele of the
cyt-1 gene encoding the cytochrome b subunit
of complex II. Mutants are hypersensitive to methyl viologen and oxidative stress, have
elevated levels of superoxide anion production, display signs of premature aging, have a
reduced lifespan, and are hypermutable (Hartman et al., 2001; Ishii et al., 1998;
Ishii et al., 2002; Senoo-Matsuda et al., 2001). A probable null mutation in the gene for the
mitochondrial leucyl-tRNA synthetase (lrs-2(mg312)) markedly
extends life span (Lee et al., 2003). The synthetase
charges mitochondrial tRNAs needed for the translation of the 12 mtDNA-encoded MRC
subunits. Mutation of the gene encoding the isopentenyl-pyrophosphate:tRNA transferase
(gro-1(e2400), abnormal growth rate), produces a Clk
(biological clock abnormal) phenotype characterized by slow development and increased
lifespan (Lemieux et al., 2001). The apparent use of
alternative translation initiation codons produces differentially targeted forms of
GRO-1; it is the loss of the mitochondria-targeted form, which presumably modifies a
subset of tRNAs and affects mitochondrial translation, that leads to the Clk phenotype.

Non-genetic interventions that affect mitochondrial function can also affect aging.
Systematic RNAi screens for increased lifespan have identified numerous mitochondrial
components, including MRC subunits, mitochondrial carrier proteins, a subunit of the
mitochondrial ribosome, and a protein involved in MRC biogenesis (Dillin et al., 2002; Lee et al., 2003). Mutations in the clk-1 gene disrupt
ubiquinone biosynthesis and significantly increase lifespan (Ewbank et al., 1997; Wong et al., 1995). Finally, exposure to antimycin A, a tight-binding inhibitor of
complex III, reduces rates of respiration and promotes longevity (Dillin et al., 2002). Given the presence of 12 key MRC genes on the
mtDNA and the intimate connections between MRC function and lifespan, it seems
inevitable that mutations that slow aging will be identified in one or more of those 12
genes.

10. Perspectives

The mitochondrial genome is often ignored although it is a vital component of animal
metabolism, physiology, and development. Few studies have specifically addressed the
C. elegans mtDNA and much of our knowledge is inferred from
investigations of vertebrate mtDNAs and from bioinformatic approaches. The C.
elegans cellular machinery required for mtDNA maintenance and expression
is almost completely unexplored; undoubtedly biological novelties remain to be exposed.
Deciphering how mitochondria communicate with the remainder of the cell will have
far-reaching implications for understanding cellular metabolism in normal and in
diseased states. Few model systems offer as many advantages as the nematode for grasping
the full importance and scope of mitochondrial genetics in animal life.

11. Acknowledgements

I wish to thank Catherine McPhalen for critical reading of the manuscript. The
Canadian Institutes of Health Research Grant MT-15336 supported this work. Bernard
Lemire is a member of the Membrane Protein Research Group, Department of Biochemistry, University of Alberta,
Edmonton, Alberta, Canada T6G 2H7.

12. References

Austin, J., and Kimble, J. (1987). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell 51, 589–599.AbstractArticle